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Clinical Chemistry 48: 121-130, 2002;
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(Clinical Chemistry. 2002;48:121-130.)
© 2002 American Association for Clinical Chemistry, Inc.

Antigen Microarrays for Serodiagnosis of Infectious Diseases

Letizia Mezzasoma1,1, Tito Bacarese-Hamilton1,1, Manlio Di Cristina1, Ruggero Rossi2, Francesco Bistoni2 and Andrea Crisanti1a

1 Department of Biology, Imperial College of Science, Technology and Medicine, London SW7 2AZ, United Kingdom.

2 Dipartimento di Medicina Clinica e Sperimentale Universitèa degli Studi di Perugia, Via del Giochetto, 006100 Perugia, Italy.

aAddress correspondence to this author at: Imperial College of Science, Technology and Medicine, Department of Biology, Biomedical Sciences Building, 5th Floor, Imperial College Road, London SW7 2AZ, United Kingdom. Fax 44-207-5945439; e-mail acrs{at}ic.ac.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Progress in robotic printing technology has allowed the development of high-density nucleic acid and protein arrays that have increased the throughput of a variety of assays. We generated protein microarrays by printing microbial antigens to simultaneously determine in human sera antibodies directed against Toxoplasma gondii, rubella virus, cytomegalovirus (CMV), and herpes simplex virus (HSV) types 1 and 2 (ToRCH antigens).

Methods: The antigens were printed on activated glass slides with high-speed robotics. The slides were incubated first with serum samples and subsequently with fluorescently labeled secondary antibodies. Human IgG and IgM bound to the printed antigens were detected by confocal scanning microscopy and quantified with internal calibration curves. Both microarrays and commercial ELISAs were utilized to detect serum antibodies against the ToRCH antigens in a panel of characterized human sera.

Results: The detection limit (mean + 2 SD) of the microarray assay was 0.5 pg of IgG or IgM bound to the slides. Within-slide, between-slide, and between-batch precision profiles showed CVs of 1.7–18% for all antigens. Overall, >80% concordance was obtained between microarray assays and ELISAs in the classification of sera; for T. gondii, CMV, and HSV1, concordance exceeded 90%.

Conclusions: The microarray is a suitable assay format for the serodiagnosis of infectious diseases and can be easily optimized for clinical use. The ToRCH assay performs equivalently to ELISA and may have potentially important advantages in throughput, convenience, and cost.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Assays with the ability to detect in parallel antibodies with different specificities have a wide range of potential applications in epidemiologic research and vaccine development as well as in the diagnosis of allergies and autoimmune and infectious diseases. Although ELISA-based tests are suitable for this purpose, available assays can be time-consuming and require large quantities of both sample and reagents, thus limiting their application for mass screening (1)(2). The advent of automated, robust microdeposition technologies has allowed the development of high-density ordered arrays of molecules (microarrays) (3). This assay format incorporates key features, such as true parallelism, miniaturization, and high throughput, that could overcome most of the current ELISA limitations (4). Studies using DNA microarrays have shown that this assay format is ideal for studying the expression profiles of thousands of genes simultaneously in tissues and organs under different experimental conditions (5)(6)(7). Currently, DNA microarrays are regarded as one of the most powerful available tools for understanding the functional relationships between the large repertoires of genes available in databases generated as a result of genome sequencing projects (8)(9).

Ordered arrays of peptides have been used for unraveling protein-protein and protein-nucleic acid interactions (10)(11). Previous reports have shown that protein arrays printed on membranes can be used to screen expression libraries for binding to target molecules as well as to unravel protein binding to either RNA or DNA (12)(13)(14)(15). More recently, arrays have been generated for high-throughput screening of recombinant antibodies (16). These antibody arrays contain thousands of bacterial clones, each expressing a different single-chain antibody. Arrays of proteins covalently bound to glass slides were shown to retain their ability to interact specifically with other proteins or with small molecules in solution (17). Protein arrays have also been used to develop comparative fluorescence assays to measure the concentrations of several specific proteins and antibodies in complex solutions (18)(19)(20)(21). These studies have shown that protein microarrays could provide a practical means to quantify thousands of different protein species in clinical or research applications. In spite of these advances, the development of protein arrays for research and clinical applications has lagged because of the poor stability of proteins, complex coupling chemistry, and weak detection signals. High-density protein arrays remain difficult to generate and to validate for clinical use.

We used a protein microarray to determine in human sera the presence of specific antibodies directed against parasite and viral antigens, including Toxoplasma gondii, cytomegalovirus (CMV), 2 rubella virus, and herpes simplex virus (HSV) types 1 and 2. The clinical performance of this assay was validated with a collection of sera previously characterized with commercial ELISAs for their reactivity against the selected microbial antigens.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
preparation of microarrays
Microbial antigens as well as human IgG and IgM at different concentrations were printed on silanized glass microscope slides (CEL Associates), using computer-controlled high-speed robotics (Total Array System; Biorobotics) (22). Samples were transferred from 384 microtiter plates to glass slides by use of stainless steel solid pins (200-µm diameter). Each pin was estimated to transfer ~1 nL of sample to the slide. Arrays consisted of a 7 x 7 matrix that included five microbial antigens printed in six replicates, the IgG and IgM internal calibration curves in duplicate, and rabbit muscle myosin as control. The fluorophores Alexa 546 and/or Alexa 594 (50 pg) were incorporated in the array as a reference signal. T. gondii, CMV, and HSV1 and HSV2 preparations were supplied by Radim S.p.A. Rubella virus antigen (grade K1S) was purchased from Microbix Biosystems Inc. Human IgG and IgM (reagent grade) were purchased from Sigma Chemical Company. Antigens were dissolved in phosphate-buffered saline [PBS; 1x (0.2 g/L KCl, 1.44 g/L Na2HPO4, 0.24 g/L KH2PO4, 8 g/L NaCl, pH 7.4)] containing Tween 20 (0.1 mL/L). Polyvinylpyrrolidone (10 g/L) was added to the human IgG and HSV1 antigen solutions, sodium dodecyl sulfate (0.1 g/L) was added to the human IgM solution, and sucrose (100 g/L) was added to the CMV antigen solution. No additives were present in the other antigen preparations. Printing was performed in a cabinet at 25 °C and 55% humidity. These conditions were constantly monitored by a thermohygrometer. Printed slides were stored for 12 h inside the cabinet before removal and storage. Slides were stored in boxes at room temperature in the presence of silica gel bags as desiccant and used within 90 days of being printed.

serum samples and elisa methods
Human sera (n = 60) were supplied by BioMedical Resources Inc. The sera were arranged in five panels on the basis of their reactivity against T. gondii, rubella virus, HSV1, HSV2, and CMV as measured by Abbott IMx assay, Sigma enzyme immunoassay (EIA), and Zeus EIA. The panels VP6627, VP6628, and VP6678 consisted of two groups of five sera containing either IgG or IgM directed against T. gondii, rubella virus, and CMV, respectively. Each panel also contained five sera selected for their lack of IgG and IgM reactivity against different ToRCH antigens. Panel VP6680 contained five sera with IgM directed against HSV1. Panel VP6679 consisted of five positive sera with IgG against HSV1 and five negative sera.

All sera were analyzed for reactivity against T. gondii, rubella virus, HSV1, HSV2, and CMV antigens by the ELISAs (EIA Well) supplied by Radim S.p.A. The assays were performed according to the manufacturer’s instructions.

processing of microarray slides
Printed slides were incubated for 1 h at room temperature with a solution containing 10 g/L bovine albumin in PBS to block nonspecific antibody binding. An adhesive tape (Gene-Frame; Abgene Limited) was used to contain samples/reagents within the array area. Serum samples were diluted 1:200 in 2x PBS containing 10 g/L bovine serum albumin and 0.1 mL/L Tween 20 and allowed to react with the array for 15 min at room temperature in a humid chamber. To reveal IgG bound to the printed antigens, the slides were washed five times (1 mL each time) with PBS containing 0.1 mL/L Tween 20 and subsequently incubated for 5 min with Alexa 546-labeled anti-human IgG monoclonal antibody 2F6 (Radim) suspended in a solution containing 2x PBS, 10 g/L bovine serum albumin, and 0.1 mL/L Tween 20. To reveal IgM, the slides were incubated for 5 min with Alexa 594-labeled goat immunoglobulins directed against the anti-human IgM µ chain (OEM Concepts Inc.). Before the fluorescence was read in the scanner, the slides were washed and dried at 37 °C. The secondary antibodies, monoclonal antibody 2F6 and the goat immunoglobulins, were labeled with succinimidyl Alexa 546 and 594, respectively, and purified according to the instructions provided by the manufacturer (Molecular Probes Inc.). The efficiency of the labeling procedure was analyzed by measuring the molar ratio of dye to protein.

data collection and analysis
The slides were analyzed with a GSI 5000 scanner. Images were generated with the ScanArrayTM software provided by GSI Lumonics and quantified using the QuantArrayTM software provided by the same company (23). Human IgG and IgM dose-response curves were fitted with a linear curve fit (Excel; Microsoft). The amounts of IgG and IgM in the sera were determined by interpolating the photomultiplier counts collected at the microbial antigen spots with the IgG and IgM internal calibration curves.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
array design and analysis of IgG and IgM calibration curves
Purified human IgG and IgM as well as T. gondii, CMV, rubella virus, HSV1, and HSV2 antigen preparations were printed on amino-silane-activated glass slides. This surface was chosen on the basis of experiments indicating that proteins, such as antibodies and albumin, bound with high efficiency and reproducibility to amino-silane-activated glass slides (data not shown). Each antigen was printed in six replicates within a 7 x 7 matrix (Fig. 1 ). The array was designed to contain a negative control (rabbit myosin) and the fluorophores Alexa 546 and Alexa 594 as sources of reference signal. Internal calibration curves were generated by printing, in duplicate, 1 nL of immunoglobulin solution containing increasing concentrations of IgG or IgM (2–50 and 0.4–8 mg/L, respectively). The reactivities of the secondary antibodies against the negative control (myosin) were used as the first point of the calibration curves. Regression analysis demonstrated that linear dose-response curves were obtained by plotting the photomultiplier counts against the amounts of immunoglobulins printed on the slides (Fig. 2 ). Both IgG and IgM calibration curves showed similar slopes and coefficients of determination (r2) close to 1. These experiments also revealed that the cross-reactivities of the anti-IgM goat serum and monoclonal antibody 2F6 against human IgG and IgM, respectively, were negligible.



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  		Figure 1. Schematic representation of the array used in this study.

Colored circles indicate the positions where T. gondii, rubella virus, CMV, HSV1, and HSV2 antigen preparations were printed in replicate. The array was designed to contain internal calibration curves generated by printing increasing amounts of purified human IgG and IgM. Rabbit myosin was printed as negative control. The fluorophores Alexa 546 and Alexa 594 were also included in the array (white circle).



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  		Figure 2. Fluorescent scans of array areas printed with increasing concentrations (top to bottom) of human IgG (A; 2, 10, 25, and 50 mg/L) and IgM (C; 0.4, 2, 4, and 8 mg/L) and incubated with anti-IgG and -IgM secondary antibodies labeled with Alexa 546 and Alexa 594, respectively.

The fluorescence was visualized in a pseudo-color scale (dark blue to white) corresponding to increasing fluorescence. The mean values of duplicate photomultiplier count (pmc) measurements were plotted as a function of the concentrations of IgG (B) and IgM (D) to generate dose-response curves. The equation and the coefficient of determination are indicated for each curve.

analysis of serum reactivity against arrayed antigens
Human sera from panels VP6627, VP6628, VP6678, VP6680, and VP6679 were used to investigate the ability of the microarray assay to reveal the presence or absence of IgG and IgM directed against the different ToRCH antigens. These sera were certified for the presence or absence of antibodies directed against distinct ToRCH antigens on the basis of the Abbott IMx assay, the Sigma EIA, or the Zeus EIA. Radim ELISAs for ToRCH antigens were used to confirm the reactivities of all sera. Fluorescent scans of microarray slides incubated with representative sera of the different panels showed that this assay allows the detection of specific antibodies directed against microbial antigens in accordance with ELISA reactivity data (Figs. 3 and 4 ). In general, sera that failed to show a positive reaction in ELISA did not react against the corresponding microbial antigens in the microarray assay, whereas fluorescent signals were usually associated with positive reactions in the ELISAs (Figs. 3 and 4 ). No reactivity was detected against the myosin negative control, thus demonstrating the absence of nonspecific interference in the assay. The r2 values for both the IgG and IgM curves in these arrays were 0.97–0.99, indicating that the internal calibration curves generated under these experimental conditions showed a high reproducibility and a linear dose-response relationship (Figs. 5 and 6 ).



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  		Figure 3. Fluorescent scans of antigen arrays incubated with samples from VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum panels, which differ according to their reactivities in ELISA in the presence of IgG directed against the ToRCH antigens.

Serum antibodies were detected by incubating the slides with Alexa 546-labeled secondary antibody directed against human IgG. The intensity of fluorescence emitted by the fluorophore was visualized in a pseudo-color scale (dark blue to white).



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  		Figure 4. Fluorescent scans of antigen arrays incubated with samples from VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum panels.

Serum antibodies were detected by incubating the slides with Alexa 594-labeled secondary antibody directed against human IgM. The fluorescence emitted by the fluorophore was visualized in a pseudo-color scale (dark blue to white).



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  		Figure 5. Mean photomultiplier count (pmc) measurements collected from the arrayed antigens (colored symbols), incubated with samples from VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum panels, were interpolated to the dose-response curves generated by plotting the fluorescent intensity as a function of printed amounts of IgG (blue diamonds).

The SE of replicate measurements was <10%. The equation and the coefficient of determination are indicated for each curve.



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  		Figure 6. Mean photomultiplier count (pmc) measurements collected from the arrayed antigens (colored symbols), incubated with samples from VP6678/1 (A), VP6678/15 (B), VP6628/2 (C), and VP6627/8 (D) serum panels, were interpolated to the dose-response curves generated by plotting the fluorescent intensity as a function of printed amounts of IgM (blue diamonds).

The SE of replicate measurements was <10%. The equation and the coefficient of determination are indicated for each curve.

A set of human sera showing different reactivities in the ELISA (low, medium, and high), was used to monitor the performance of the microarray assay for each ToRCH antigen. This analysis revealed that increasing concentrations of IgG directed against the microbial antigens generated fluorescence signals with precision profiles that showed in most cases a CV <10% within and between slides (Table 1 ). Slightly higher CV values were observed when comparing slides from different batches (Table 1 ). These values were similar to ELISA-generated data, as stated in the instruction manual for the ELISA. The detection limit of the microarray assay was determined by assessing the reactivity of the sera against 20 replicates of rabbit myosin printed on slides. The detection limit, defined as mean photomultiplier counts of the negative control plus 2 SD interpolated on the IgG calibration curve, was 0.5 pg.


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  		Table 1. Within-slide, between-slide, and between-batch precision profiles of the microarray assay.

microarray assay for multianalyte serodiagnosis
We compared the microarray assay and Radim ELISA for their ability to determine in human sera the presence or absence of IgG reactive against the ToRCH antigens. This analysis could not be extended to serum IgM because we lacked a sufficient number of characterized positive sera for accurate comparison with the ELISA. Notably, the antigen microarray and the Radim ELISA shared the same antigen and antibody reagents, facilitating the comparison of the two diagnostic assays. The cutoff values of the distinct serum reactivity determinations within the microarray assay were calculated using reference sera that, according to information that accompanied the Abbot IMx, Sigma EIA, and Radim ELISA, lacked antibodies directed against T. gondii, rubella virus, CMV, HSV1, or HSV2. These values incorporated the 95th percentile of these reference sera. The reactivities of the serum samples were classified as either positive or negative according to the presence of IgG concentrations above or below the cutoff value. Sera with IgG concentrations between ± 10% of the cutoff value were regarded as equivocal. According to these criteria, the microarray assay classified 19.6% of samples as positive, 76.8% as negative, and 3.6% as equivocal for the presence of IgG directed against T. gondii; corresponding ELISA data were 21.4% positive, 69.7% negative, and 8.9% equivocal (Table 2 ). For CMV, the microarray classified 64.3% of samples as positive, 32.1% as negative, and 3.6% as equivocal; corresponding ELISA data were 66.1% positive, 32.1% negative, and 1.8% equivocal. For rubella virus the microarray classified 17.9% of samples as positive, 75% as negative, and 7.1% as equivocal; ELISA classified 87.5% as positive, 1.8% as negative, and 10.7% as equivocal (Table 2 ).


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  		Table 2. Comparison of microarray and ELISA for their ability to detect IgG directed against ToRCH antigens in human sera.

For HSV1, the microarray classified 80.3% of samples as positive, 17.9% as negative, and 1.8% as equivocal; corresponding ELISA data were 78.6% positive, 19.6% negative, and 1.8% equivocal. For HSV2, the microarray classified 22.2% of samples as positive, 66.7% as negative, and 11.1% as equivocal; corresponding ELISA data were 18.5% positive, 51.9% negative, and 29.6% equivocal (Table 2Up ).

comparison of test protocols
Comparison of microarray assay and ELISA test protocols (Table 3 ) indicated that the major advantage of the microarray test is that regardless of the number of analytes determined on a slide, the time-to-result and costs were the same. With ELISA formats, each analyte is determined in a separate assay, thereby increasing time and costs.


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  		Table 3. Comparison of microarray and ELISA test protocols.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data indicate that a protein microarray assay with indirect fluorescence detection can be used to determine the presence or absence of specific antibodies directed against various microbial antigens in human sera. The presence of internal dose-response IgG and IgM calibration curves allowed us to demonstrate that antibodies from different serum samples bound to printed antigens can be quantified with high reproducibility. In addition, the calibration curves provided unique advantages over current immunoassay protocols in which the calibration curves and the samples are processed in separate tubes or wells. In these assays, matrix problems (differences between the calibrator and sample matrices) occur frequently and represent a known source of bias (24). In contrast, in our microarray format any given signal developed within the arrayed antigens is interpolated to a calibration curve processed under the same physicochemical conditions (e.g., time, temperature, protein content, fat content, and bilirubin concentration) as the antibody reactivity being determined. Under these conditions, the dose-response curve will compensate for variability in the sample physicochemical conditions, thus minimizing matrix effects.

The use of a dedicated microarray robot coupled with optimized printing solutions and glass substrates allowed us to generate high-quality fluorescence data suitable for a study aimed at assessing the clinical performance of the ToRCH microarray assay. For this purpose the microarray assay and several commercially available ELISAs were compared for their ability to reveal IgG directed against the selected microbial antigens in a set of characterized human sera. Notably, both the microarray assay and the ELISAs used in the comparative study shared the same antigen preparations and antibody reagents. This analysis revealed that the microarray assay is able to identify positive and negative sera with the same efficiency as the ELISAs. For rubella, the Radim ELISA classified 49 samples (87.5%) as positive, whereas only 10 (17.9%) of these sera were shown to have specific antibodies in the microarray assay. A small number of these samples (n = 10) were analyzed in an independent assay (Abbott IMx), and the results of this control experiment agreed with the microarray assay rather than with the Radim ELISA data. Our data also indicated that the microarray assay could be used to determine the presence or absence of specific serum IgM. The number of positive sera available was too small to infer conclusions concerning the clinical performance, but this preliminary analysis indicated that similar results were obtained with ELISAs and the microarray assay.

In conclusion, our findings demonstrate that not only can good analytical and clinical data be obtained with microarrays, but that this test format may have potentially important advantages in convenience and cost compared with standard ELISA formats. The microarray test format can be incorporated in a fully automated random-access immunoassay platform for routine use in clinical chemistry laboratories. Protein microarrays have the potential to improve, in the near future, the diagnosis of infectious diseases and pathologic conditions such as allergies and autoimmune diseases.


   Acknowledgments
 
L. Mezzasoma was supported by the University of Perugia. This work was entirely funded by a grant by Radim S.p.A. (Rome, Italy). We thank Clare McBrearty and Stanislavo Marcolini for excellent technical assistance and Luigi Sparano for encouragement and support.


   Footnotes
 
1 These authors contributed equally to this work.

2 Nonstandard abbreviations: CMV, cytomegalovirus; HSV, herpes simplex virus; PBS, phosphate-buffered saline; and EIA, enzyme immunoassay.


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